Week 13
112812
- Concentrated the protein after FPLC to 1ml.
- the concentration got higher. Guess it worked!
- Gel image of our protein characterization
Lane 1: n/a
Lane 2: ladder
Lane 3: sample 1
Lane 4: n/a
Lane 5: sample 3
Lane 6: sample 4
Lane 7: n/a
Lane 8: elution 1
Lane 9: elution 2
Lane 10: n/a
Week 12
112612 - Good. Dr B
- Did Finished our protein expression/purification/characterizaiton.
- Finished FPLC and concentrated protein is stored.
Here is the NanoDrop result right after FPLC without concentration.
Week 11
Tom e- include some data or results here:--Dr. B 11/19/12
- Continue on Homology model.
- Start on M&M cloning protocol
Week 10
- Protein expression protocol started. (FtHAP enzyme surrogate)
- Homology model
<span style="background-color: #ffffff; font-size: 12px;">[[#B|1auiA]]
><span style="color: #152c57; text-decoration: none;">[[@http://swissmodel.expasy.org/workspace/smtl/index.php?method=results&wt=1&pdbid=1auiA|[Template]]]</span>|1auiA|2.1| HUMAN CALCINEURIN HETERODIMER
Length = 378
<span style="color: #152c57; text-decoration: none;">[[http://swissmodel.expasy.org/workspace/index.php?func=getFile&file=getTeIdentProjectFile&userid=tomjonggu@gmail.com&prjid=P000001&token=f0052b77db8ae4f80053b1c43f4680c1&key=58899f5b0b1f0c9ae9b05efda02a5915&M=B&V=0&N=1auiA|[Display Alignment in DeepView]]]</span>
Score = 336 bits (861), Expect = 2e-92, Method: Composition-based stats.
Identities = 165/372 (44%), Positives = 234/372 (62%), Gaps = 26/372 (6%)
Query: 17 PPPLWCEAKREALIDGKGKVNLEVMLVHFLRQGRLSKHDALNIIQNASSVLRTEPNVLRI 76
PP AK + D GK ++++ H +++GRL + AL II +S+LR E N+L I
Sbjct: 11 PPSHRLTAKE--VFDNDGKPRVDILKAHLMKEGRLEESVALRIITEGASILRQEKNLLDI 68
Query: 77 ADPSVVVVGDVRGQFYDLAKIIFIGNMFSRAKT-YLFLGNYIDSCFFSTECILLLLAAKL 135
D V V GD+ GQF+DL K+ +G S A T YLFLG+Y+D +FS EC+L L A K+
Sbjct: 69 -DAPVTVCGDIHGQFFDLMKLFEVGG--SPANTRYLFLGDYVDRGYFSIECVLYLWALKI 125
Query: 136 THPSCVFLLRGNHECRFMSNIFDFRGECLKKYNDDVYEAIMTAFDCLPLAAVVNNQYFCV 195
+P +FLLRGNHECR ++ F F+ EC KY++ VY+A M AFDCLPLAA++N Q+ CV
Sbjct: 126 LYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCV 185
Query: 196 HGGLSPDVTSVDNIRLIYRFREPPSKGAMCDLLWSDPFWDVENPSSVCEGSRDDYYTPGN 255
HGGLSP++ ++D+IR + RF+EPP+ G MCD+LWSDP D N
Sbjct: 186 HGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEK--------------- 230
Query: 256 GPSYGTAPCFLDNEQRGCSYLFNYHSVKHFLLTNGLLCVVRSHEVQDDGYKLYRFNSVSN 315
T F N RGCSY ++Y +V FL N LL ++R+HE QD GY++YR + +
Sbjct: 231 -----TQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTG 285
Query: 316 FPCMMSVFSAPNYCDNLHNKGAVLILEGKQIGIKQFCCSPHPYVLPKHLNAFEWSFSYLL 375
FP ++++FSAPNY D +NK AVL E + I+QF CSPHPY LP ++ F WS ++
Sbjct: 286 FPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVFTWSLPFVG 345
Query: 376 DSVRDIFASIIS 387
+ V ++ ++++
Sbjct: 346 EKVTEMLVNVLN 357</span>
The QMEAN4 score is a composite score consisting of a linear combination of 4 statistical potential terms (estimated model reliability between 0-1). The pseudo-energies of the contributing terms are given below together with their Z-scores with respect to scores obtained for high-resolution experimental structures of similar size solved by X-ray crystallography:
Scoring function term
Raw score
Z-score
C_beta interaction energy
-68.40
-1.48
All-atom pairwise energy
-5657.95
-2.05
Solvation energy
-7.57
-2.81
Torsion angle energy
-51.94
-2.40
QMEAN4 score
0.539
-3.77
If you publish results from QMEAN, please cite the following paper: Benkert P, Biasini M, Schwede T. (2011). "Toward the estimation of the absolute quality of individual protein structure models." Bioinformatics, 27(3):343-50.
Local Model Quality Estimation: Anolea / QMEAN / Gromos:
References If you publish results using SWISS-MODEL, please cite the following papers: - Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 22,195-201. - Schwede T, Kopp J, Guex N, and Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Research 31: 3381-3385. - Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling.Electrophoresis 18: 2714-2723.
Week 9
102912 - Re-checked my gel for primary PCR with both 56 and 57 degree celcius
Lane 1: skipped
Lane 2: 100bp buffer
Lane 3-7: N/A
Lane 8: 57 degree celcius Primary PCR
Lane 9: 56 degree celcius Primary PCR
Week 8
102512- Finished my research report.
102612- Ran the Gel of my 2nd attempt of PCR for my target protein \
Lane 1: skipped
Lane 2: skipped
Lane 3: 100 bp ladder
Lane 4: N/A
Lane 5: primary PCR
Lane 6: Secondary PCR
: this time i lowered all the temperatures of all cycles. Messed up result... will try again with only 1 and 2 degree celcius down on annealing temperature.
102712- Ran my 3rd attempt of PCR primary with 57 and 56 degree celsius for annealing temperature.
Apparently... the Gel did not contain any etBr... gonna run it again.
Week 7
102112 - Tom - ok - but show your gel images. -- Dr. B
101712- Did my Virtual Refresher. I started this on Monday and just ran the 2nd job. Hope to get a result by tomorrow to analyze.
There are the top 10 ligands
101712- Also, i redid my primary PCR. I lowered the melting temperature by 1 degree celcius just to prevent any denaturation. I will run the gel for this and see if i get any result and will either keep going on or find out what's wrong with this.
Week 6
101612 - tom, ok try to figure out what is going on here and see if you can get Primary and Secondary to work. -- Dr. B
I think we should sit down and double check the groups primer design to make sure there aren't any errors there.
100812- Started my primary PCR for my target. Sajan already made a oligo-mix for our protein (phosphatase 2B) so i used the sample.
101012- did my secondary PCR for the sample, waiting for the result
101212- Finished my 2nd PCR and have a gel image. The gel had Aldo and Brandon's sample also that im not sure if my PCR worked.
Week 5
100912 - Tom show some wet lab results too. DR. B
-Did my PyMol refresher.
PyMol Refresher
Images.
Figure 1. Image of 2H2Q molecule. Substrate is in red, hydrophobic with pink, ionic with blue, and polar residues w ith blue.
Figure 2. Image of 3CL9 molecule. Inhibitor (MTX) is in pink, substrate with cyan, and active site with orange.
Figure 3. 1U72 molecule image. Cyan is 3CL9 and green is 1U72. Orange is active site of 1U72 and blue is active site of 3CL9.
The RMS value was 1.126. The enzymes could be differentially targeted with a single drug since the binding sites for both MTX in the molecules are similar. The active site was mostly overlapping except that 1u72 had asp but 3cl9 did not have them in the active site.
Figure 4. BLAST result of 3CL9 and 1U72
Figure 5. Image of 3HBB molecule. The inhibitor (TMQ) is in pink, polar contacts with black, active site with orange. The binding mode of TMQ to T. cruzi DHFR-TS is not significantly different from that of MTX to human DHFR from 1u72.
Week 4
093012- Tom, good - label this gel a little better so that I know what the bands are. -- Dr. B
Had 3 exams this week, so my lab effort was a little bit less devoted, forgive me coach.
- Did 2nd PCR today (9.26/12) and left it at the lab for running. Will check the gel tomorrow for confirmation.
failed PCR 2.....
- Did PCR primer design for pNIC - Bsa4 cloning The DNA sequence # 1 is:
Week 3
Tom - ok not sure your amplification on PCR worked. Try again and check that your cycling protocol is correct. Good jobincluding the ladder image on right. Try to crop the main image though. Nice job on Midiprep. -- Dr. B
My 1st PCR was thrown away by someone, so I will have to re-do this lab on friday.
Re-did my 1st PCR.
Not sure if that is the right one though..
Midiprep was done today. The result Nanodrop image is provided below.
The sample was initially combined into one tube, so it might have been too concentrated.
Week 1 & 2
Other labs were done this week. Primer dilution, DNA sequence analysis (which is uploaded in the google docs).
somehow, my gel image can't be converted to jpeg... i dont know how to do that
Restriction Digest of pGBR22
Lane 1; Skip
Lane 2; 1 kb NEB DNA Ladder
Lane 3; Uncut pGBR22 plasmid
Lane 4; EcoRI-Hf
Lane 5; PvuII
Lane 6; EcoRI-Hf/PvuII
Personal target page:
Someone deleted my original target page so i had to generate another target page. It was about T. Brucei Here is the basic information about the target.
- Did enzyme and inhibition assay.
Enzyme essay result:
Inhibition assay
Week 13
112812
- Concentrated the protein after FPLC to 1ml.
- the concentration got higher. Guess it worked!
- Gel image of our protein characterization
Lane 1: n/a
Lane 2: ladder
Lane 3: sample 1
Lane 4: n/a
Lane 5: sample 3
Lane 6: sample 4
Lane 7: n/a
Lane 8: elution 1
Lane 9: elution 2
Lane 10: n/a
Week 12
112612 - Good. Dr B
- Did Finished our protein expression/purification/characterizaiton.
- Finished FPLC and concentrated protein is stored.
Here is the NanoDrop result right after FPLC without concentration.
Week 11
Tom e- include some data or results here:--Dr. B 11/19/12
- Continue on Homology model.
- Start on M&M cloning protocol
Week 10
- Protein expression protocol started. (FtHAP enzyme surrogate)
- Homology model
<span style="background-color: #ffffff; font-size: 12px;">[[#B|1auiA]] ><span style="color: #152c57; text-decoration: none;">[[@http://swissmodel.expasy.org/workspace/smtl/index.php?method=results&wt=1&pdbid=1auiA|[Template]]]</span>|1auiA|2.1| HUMAN CALCINEURIN HETERODIMER Length = 378 <span style="color: #152c57; text-decoration: none;">[[http://swissmodel.expasy.org/workspace/index.php?func=getFile&file=getTeIdentProjectFile&userid=tomjonggu@gmail.com&prjid=P000001&token=f0052b77db8ae4f80053b1c43f4680c1&key=58899f5b0b1f0c9ae9b05efda02a5915&M=B&V=0&N=1auiA|[Display Alignment in DeepView]]]</span> Score = 336 bits (861), Expect = 2e-92, Method: Composition-based stats. Identities = 165/372 (44%), Positives = 234/372 (62%), Gaps = 26/372 (6%) Query: 17 PPPLWCEAKREALIDGKGKVNLEVMLVHFLRQGRLSKHDALNIIQNASSVLRTEPNVLRI 76 PP AK + D GK ++++ H +++GRL + AL II +S+LR E N+L I Sbjct: 11 PPSHRLTAKE--VFDNDGKPRVDILKAHLMKEGRLEESVALRIITEGASILRQEKNLLDI 68 Query: 77 ADPSVVVVGDVRGQFYDLAKIIFIGNMFSRAKT-YLFLGNYIDSCFFSTECILLLLAAKL 135 D V V GD+ GQF+DL K+ +G S A T YLFLG+Y+D +FS EC+L L A K+ Sbjct: 69 -DAPVTVCGDIHGQFFDLMKLFEVGG--SPANTRYLFLGDYVDRGYFSIECVLYLWALKI 125 Query: 136 THPSCVFLLRGNHECRFMSNIFDFRGECLKKYNDDVYEAIMTAFDCLPLAAVVNNQYFCV 195 +P +FLLRGNHECR ++ F F+ EC KY++ VY+A M AFDCLPLAA++N Q+ CV Sbjct: 126 LYPKTLFLLRGNHECRHLTEYFTFKQECKIKYSERVYDACMDAFDCLPLAALMNQQFLCV 185 Query: 196 HGGLSPDVTSVDNIRLIYRFREPPSKGAMCDLLWSDPFWDVENPSSVCEGSRDDYYTPGN 255 HGGLSP++ ++D+IR + RF+EPP+ G MCD+LWSDP D N Sbjct: 186 HGGLSPEINTLDDIRKLDRFKEPPAYGPMCDILWSDPLEDFGNEK--------------- 230 Query: 256 GPSYGTAPCFLDNEQRGCSYLFNYHSVKHFLLTNGLLCVVRSHEVQDDGYKLYRFNSVSN 315 T F N RGCSY ++Y +V FL N LL ++R+HE QD GY++YR + + Sbjct: 231 -----TQEHFTHNTVRGCSYFYSYPAVCEFLQHNNLLSILRAHEAQDAGYRMYRKSQTTG 285 Query: 316 FPCMMSVFSAPNYCDNLHNKGAVLILEGKQIGIKQFCCSPHPYVLPKHLNAFEWSFSYLL 375 FP ++++FSAPNY D +NK AVL E + I+QF CSPHPY LP ++ F WS ++ Sbjct: 286 FPSLITIFSAPNYLDVYNNKAAVLKYENNVMNIRQFNCSPHPYWLPNFMDVFTWSLPFVG 345 Query: 376 DSVRDIFASIIS 387 + V ++ ++++ Sbjct: 346 EKVTEMLVNVLN 357</span>Welcome tomjonggu@gmail.com* Documentation
Print/Save this page as
Model Summary
|| ||
Only user specified template was used for modelling.
Model built :SINGLE CHAIN
Ligands in the model: none.
display model: as [pdb] - as [DeepView project] - in
download model: as [pdb] - as [Deepview project] - as [text]
Global Model Quality Estimation
|||||| QMEAN4 global scores:
Z-Score: -3.77
Plot 1: [save png]
Plot 2: [save png]
[save png]
[save png]
[save jpg] [save pdb]
[save raw scores]
Benkert P, Biasini M, Schwede T. (2011). "Toward the estimation of the absolute quality of individual protein structure models." Bioinformatics, 27(3):343-50.
Local Model Quality Estimation: Anolea / QMEAN / Gromos:
Download raw data: [Anolea Data] [QMEAN Data]
Alignment
Modelling Log
Template Selection Log
Quaternary Structure Modeling Log
Ligand Modeling Log
References
If you publish results using SWISS-MODEL, please cite the following papers:
- Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 22,195-201.
- Schwede T, Kopp J, Guex N, and Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Research 31: 3381-3385.
- Guex, N. and Peitsch, M. C. (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modelling.Electrophoresis 18: 2714-2723.
Week 9
102912 - Re-checked my gel for primary PCR with both 56 and 57 degree celcius
Lane 1: skipped
Lane 2: 100bp buffer
Lane 3-7: N/A
Lane 8: 57 degree celcius Primary PCR
Lane 9: 56 degree celcius Primary PCR
Week 8
102512- Finished my research report.
102612- Ran the Gel of my 2nd attempt of PCR for my target protein
Lane 1: skipped
Lane 2: skipped
Lane 3: 100 bp ladder
Lane 4: N/A
Lane 5: primary PCR
Lane 6: Secondary PCR
: this time i lowered all the temperatures of all cycles. Messed up result... will try again with only 1 and 2 degree celcius down on annealing temperature.
102712- Ran my 3rd attempt of PCR primary with 57 and 56 degree celsius for annealing temperature.
Apparently... the Gel did not contain any etBr... gonna run it again.
Week 7
102112 - Tom - ok - but show your gel images. -- Dr. B
101712- Did my Virtual Refresher. I started this on Monday and just ran the 2nd job. Hope to get a result by tomorrow to analyze.
There are the top 10 ligands
101712- Also, i redid my primary PCR. I lowered the melting temperature by 1 degree celcius just to prevent any denaturation. I will run the gel for this and see if i get any result and will either keep going on or find out what's wrong with this.
Week 6
101612 - tom, ok try to figure out what is going on here and see if you can get Primary and Secondary to work. -- Dr. B
I think we should sit down and double check the groups primer design to make sure there aren't any errors there.
100812- Started my primary PCR for my target. Sajan already made a oligo-mix for our protein (phosphatase 2B) so i used the sample.
101012- did my secondary PCR for the sample, waiting for the result
101212- Finished my 2nd PCR and have a gel image. The gel had Aldo and Brandon's sample also that im not sure if my PCR worked.
Week 5
100912 - Tom show some wet lab results too. DR. B
-Did my PyMol refresher.
PyMol Refresher
Images.
Figure 1. Image of 2H2Q molecule. Substrate is in red, hydrophobic with pink, ionic with blue, and polar residues w ith blue.
Figure 2. Image of 3CL9 molecule. Inhibitor (MTX) is in pink, substrate with cyan, and active site with orange.
Figure 3. 1U72 molecule image. Cyan is 3CL9 and green is 1U72. Orange is active site of 1U72 and blue is active site of 3CL9.
The RMS value was 1.126. The enzymes could be differentially targeted with a single drug since the binding sites for both MTX in the molecules are similar. The active site was mostly overlapping except that 1u72 had asp but 3cl9 did not have them in the active site.
Figure 4. BLAST result of 3CL9 and 1U72
Figure 5. Image of 3HBB molecule. The inhibitor (TMQ) is in pink, polar contacts with black, active site with orange. The binding mode of TMQ to T. cruzi DHFR-TS is not significantly different from that of MTX to human DHFR from 1u72.
Week 4
093012- Tom, good - label this gel a little better so that I know what the bands are. -- Dr. B
Had 3 exams this week, so my lab effort was a little bit less devoted, forgive me coach.
- Did 2nd PCR today (9.26/12) and left it at the lab for running. Will check the gel tomorrow for confirmation.
failed PCR 2.....
- Did PCR primer design for pNIC - Bsa4 cloning
The DNA sequence # 1 is:
----------------------------------------------------------------
1 ATGCTGCCAATCCTGAAAGCGGGTGAAGACGAGGGTGGTCCGCGTATCCCGCCACCTCTG
61 TGGTGCGAAGCGAAACGTGAAGCGCTGATCGACGGTAAAGGTAAAGTTAACCTGGAAGTC
121 ATGCTGGTTCACTTCCTCCGCCAGGGTCGTCTGTCTAAACACGACGCGCTGAACATCATC
181 CAAAATGCATCTTCCGTACTGCGTACCGAACCGAACGTCCTGCGTATCGCCGATCCGTCT
241 GTTGTTGTCGTTGGTGACGTACGCGGCCAATTCTATGACCTGGCGAAAATCATCTTCATC
301 GGTAATATGTTCTCTCGCGCCAAAACCTACCTGTTCCTGGGTAACTACATCGACTCTTGC
361 TTCTTCTCTACCGAGTGCATCCTCCTCCTGCTGGCGGCGAAGCTGACCCATCCTTCTTGC
421 GTATTCCTGCTCCGTGGCAACCACGAATGCCGTTTCATGAGCAACATTTTTGACTTTCGC
481 GGTGAATGCCTGAAGAAATACAACGACGACGTTTACGAGGCGATCATGACCGCGTTCGAC
541 TGCCTGCCGCTGGCCGCTGTCGTTAACAACCAATACTTCTGCGTTCACGGTGGCCTGTCC
601 CCGGACGTTACGTCTGTGGACAACATTCGTCTGATCTACCGTTTCCGCGAACCGCCTTCT
661 AAGGGTGCGATGTGTGATCTGCTGTGGAGCGACCCGTTCTGGGACGTTGAAAACCCGTCT
721 TCTGTTTGTGAAGGCTCTCGTGACGACTACTACACCCCAGGCAATGGTCCGTCTTACGGT
781 ACGGCGCCATGCTTCCTGGATAATGAACAACGCGGCTGTAGCTATCTGTTCAACTACCAC
841 TCCGTCAAACACTTTCTGCTCACTAACGGTCTGCTCTGTGTTGTTCGTTCTCACGAAGTT
901 CAGGACGACGGTTACAAACTGTATCGTTTTAACTCCGTGTCTAACTTTCCGTGCATGATG
961 TCTGTTTTCTCTGCGCCTAACTACTGCGACAACCTGCATAACAAAGGCGCGGTTCTGATC
1021 CTGGAGGGTAAACAGATCGGTATCAAACAGTTCTGCTGCTCCCCGCACCCGTACGTTCTG
1081 CCGAAACACCTCAACGCCTTCGAATGGTCTTTCAGCTACCTCCTGGACTCTGTTCGCGAC
1141 ATCTTCGCGTCTATTATCTCTTGCGAAGGTGCGTAA
----------------------------------------------------------
Filtered:
ATGCTGCCAATCCTGAAAGCGGGTGAAGACGAGGGTGGTCCGCGTATCCCGCCACCTCTG TGGTGCGAAGCGAAACGTGAAGCGCTGATCGACGGTAAAGGTAAAGTTAACCTGGAAGTC ATGCTGGTTCACTTCCTCCGCCAGGGTCGTCTGTCTAAACACGACGCGCTGAACATCATC CAAAATGCATCTTCCGTACTGCGTACCGAACCGAACGTCCTGCGTATCGCCGATCCGTCT GTTGTTGTCGTTGGTGACGTACGCGGCCAATTCTATGACCTGGCGAAAATCATCTTCATC GGTAATATGTTCTCTCGCGCCAAAACCTACCTGTTCCTGGGTAACTACATCGACTCTTGC TTCTTCTCTACCGAGTGCATCCTCCTCCTGCTGGCGGCGAAGCTGACCCATCCTTCTTGC GTATTCCTGCTCCGTGGCAACCACGAATGCCGTTTCATGAGCAACATTTTTGACTTTCGC GGTGAATGCCTGAAGAAATACAACGACGACGTTTACGAGGCGATCATGACCGCGTTCGAC TGCCTGCCGCTGGCCGCTGTCGTTAACAACCAATACTTCTGCGTTCACGGTGGCCTGTCC CCGGACGTTACGTCTGTGGACAACATTCGTCTGATCTACCGTTTCCGCGAACCGCCTTCT AAGGGTGCGATGTGTGATCTGCTGTGGAGCGACCCGTTCTGGGACGTTGAAAACCCGTCT TCTGTTTGTGAAGGCTCTCGTGACGACTACTACACCCCAGGCAATGGTCCGTCTTACGGT ACGGCGCCATGCTTCCTGGATAATGAACAACGCGGCTGTAGCTATCTGTTCAACTACCAC TCCGTCAAACACTTTCTGCTCACTAACGGTCTGCTCTGTGTTGTTCGTTCTCACGAAGTT CAGGACGACGGTTACAAACTGTATCGTTTTAACTCCGTGTCTAACTTTCCGTGCATGATG TCTGTTTTCTCTGCGCCTAACTACTGCGACAACCTGCATAACAAAGGCGCGGTTCTGATC CTGGAGGGTAAACAGATCGGTATCAAACAGTTCTGCTGCTCCCCGCACCCGTACGTTCTG CCGAAACACCTCAACGCCTTCGAATGGTCTTTCAGCTACCTCCTGGACTCTGTTCGCGAC ATCTTCGCGTCTATTATCTCTTGCGAAGGTGCGTAA
Forward sequence:
TACTTCCAATCCATGCTGCCAATCCTGAAAGCG
Reverse sequence:
TATCCACCTTTACTGTTACGCACCTTCGCAAGA
Forward primer:
TACTTCCAATCCATGCTGCCAATCCTGAAAGCG
33 bp, GC content: 48.5%
Mg2+ temperature
0mM: 65 °C, 1.5mM: 72°C, 2mM: 72.5°C, 4mM: 73.4°C, 6mM: 73.9°C
Reverse primer:
TATCCACCTTTACTG.TTACGCACCTTCGCAAGA
33 bp, GC content: 45.5%
Mg2+ temperature
0mM: 63.8°C, 1.5mM: 71°C, 2mM: 71.5°C, 4mM: 72.5°C, 6mM: 73°C
Inserted version:
ATGCTGCCAATCCTGAAAGCGGGTGAAGACGAGGGTGGTCCGCGTATCCCGCCACCTCTG TGGTGCGAAGCGAAACGTGAAGCGCTGATCGACGGTAAAGGTAAAGTTAACCTGGAAGTC ATGCTGGTTCACTTCCTCCGCCAGGGTCGTCTGTCTAAACACGACGCGCTGAACATCATC CAAAATGCATCTTCCGTACTGCGTACCGAACCGAACGTCCTGCGTATCGCCGATCCGTCT GTTGTTGTCGTTGGTGACGTACGCGGCCAATTCTATGACCTGGCGAAAATCATCTTCATC GGTAATATGTTCTCTCGCGCCAAAACCTACCTGTTCCTGGGTAACTACATCGACTCTTGC TTCTTCTCTACCGAGTGCATCCTCCTCCTGCTGGCGGCGAAGCTGACCCATCCTTCTTGC GTATTCCTGCTCCGTGGCAACCACGAATGCCGTTTCATGAGCAACATTTTTGACTTTCGC GGTGAATGCCTGAAGAAATACAACGACGACGTTTACGAGGCGATCATGACCGCGTTCGAC TGCCTGCCGCTGGCCGCTGTCGTTAACAACCAATACTTCTGCGTTCACGGTGGCCTGTCC CCGGACGTTACGTCTGTGGACAACATTCGTCTGATCTACCGTTTCCGCGAACCGCCTTCT AAGGGTGCGATGTGTGATCTGCTGTGGAGCGACCCGTTCTGGGACGTTGAAAACCCGTCT TCTGTTTGTGAAGGCTCTCGTGACGACTACTACACCCCAGGCAATGGTCCGTCTTACGGT ACGGCGCCATGCTTCCTGGATAATGAACAACGCGGCTGTAGCTATCTGTTCAACTACCAC TCCGTCAAACACTTTCTGCTCACTAACGGTCTGCTCTGTGTTGTTCGTTCTCACGAAGTT CAGGACGACGGTTACAAACTGTATCGTTTTAACTCCGTGTCTAACTTTCCGTGCATGATG TCTGTTTTCTCTGCGCCTAACTACTGCGACAACCTGCATAACAAAGGCGCGGTTCTGATC CTGGAGGGTAAACAGATCGGTATCAAACAGTTCTGCTGCTCCCCGCACCCGTACGTTCTG CCGAAACACCTCAACGCCTTCGAATGGTCTTTCAGCTACCTCCTGGACTCTGTTCGCGAC ATCTTCGCGTCTATTTCTTGCGAAGGTGCGTAACAGTAAAGGTGGATA
pNIC Bsa4 FASTA
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATATGCTGCCAATCCTGAAAGCGGGTGAAGACGAGGGTGGTCCGCGTATCCCGCCACCTCTGTGGTGCGAAGCGAAACGTGAAGCGCTGATCGACGGTAAAGGTAAAGTTAACCTGGAAGTCATGCTGGTTCACTTCCTCCGCCAGGGTCGTCTGTCTAAACACGACGCGCTGAACATCATCCAAAATGCATCTTCCGTACTGCGTACCGAACCGAACGTCCTGCGTATCGCCGATCCGTCT GTTGTTGTCGTTGGTGACGTACGCGGCCAATTCTATGACCTGGCGAAAATCATCTTCATC GGTAATATGTTCTCTCGCGCCAAAACCTACCTGTTCCTGGGTAACTACATCGACTCTTGC TTCTTCTCTACCGAGTGCATCCTCCTCCTGCTGGCGGCGAAGCTGACCCATCCTTCTTGC GTATTCCTGCTCCGTGGCAACCACGAATGCCGTTTCATGAGCAACATTTTTGACTTTCGC GGTGAATGCCTGAAGAAATACAACGACGACGTTTACGAGGCGATCATGACCGCGTTCGAC TGCCTGCCGCTGGCCGCTGTCGTTAACAACCAATACTTCTGCGTTCACGGTGGCCTGTCC CCGGACGTTACGTCTGTGGACAACATTCGTCTGATCTACCGTTTCCGCGAACCGCCTTCT AAGGGTGCGATGTGTGATCTGCTGTGGAGCGACCCGTTCTGGGACGTTGAAAACCCGTCT TCTGTTTGTGAAGGCTCTCGTGACGACTACTACACCCCAGGCAATGGTCCGTCTTACGGT ACGGCGCCATGCTTCCTGGATAATGAACAACGCGGCTGTAGCTATCTGTTCAACTACCAC TCCGTCAAACACTTTCTGCTCACTAACGGTCTGCTCTGTGTTGTTCGTTCTCACGAAGTT CAGGACGACGGTTACAAACTGTATCGTTTTAACTCCGTGTCTAACTTTCCGTGCATGATG TCTGTTTTCTCTGCGCCTAACTACTGCGACAACCTGCATAACAAAGGCGCGGTTCTGATC CTGGAGGGTAAACAGATCGGTATCAAACAGTTCTGCTGCTCCCCGCACCCGTACGTTCTG CCGAAACACCTCAACGCCTTCGAATGGTCTTTCAGCTACCTCCTGGACTCTGTTCGCGAC ATCTTCGCGTCTATTTCTTGCGAAGGTGCGTAACAGTAAAGGTGGATACGGATCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAAT
Week 3
Tom - ok not sure your amplification on PCR worked. Try again and check that your cycling protocol is correct. Good jobincluding the ladder image on right. Try to crop the main image though. Nice job on Midiprep. -- Dr. B
My 1st PCR was thrown away by someone, so I will have to re-do this lab on friday.
Re-did my 1st PCR.
Not sure if that is the right one though..
Midiprep was done today. The result Nanodrop image is provided below.
The sample was initially combined into one tube, so it might have been too concentrated.
Week 1 & 2
Other labs were done this week. Primer dilution, DNA sequence analysis (which is uploaded in the google docs).
somehow, my gel image can't be converted to jpeg... i dont know how to do that
Restriction Digest of pGBR22
Lane 1; Skip
Lane 2; 1 kb NEB DNA Ladder
Lane 3; Uncut pGBR22 plasmid
Lane 4; EcoRI-Hf
Lane 5; PvuII
Lane 6; EcoRI-Hf/PvuII
Personal target page:
Someone deleted my original target page so i had to generate another target page.
It was about T. Brucei
Here is the basic information about the target.
Disease: Trypansoma brucei
Enzyme: Protein Phosphatase 2B
Divya - you need your gene ID info and protein ID info here. -- Dr. B
Essentiality: Essential, used for cytokinesis
Not complex
EC#: 3.1.3.16
Drugability: 0.8 (yes)
Assay:
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/calcineurin.Par.0001.File.tmp/calcineurin.pdf
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
crystal structure:
**Amino Acid sequence:**
length: 391
MLPILKAGEDEGGPRIPPPLWCEAKREALIDGKGKVNLEVMLVHFLRQGRLSKHDALNIIQNASSVLRTE PNVLRIADPSVVVVGDVRGQFYDLAKIIFIGNMFSRAKTYLFLGNYIDSCFFSTECILLLLAAKLTHPSC VFLLRGNHECRFMSNIFDFRGECLKKYNDDVYEAIMTAFDCLPLAAVVNNQYFCVHGGLSPDVTSVDNIR LIYRFREPPSKGAMCDLLWSDPFWDVENPSSVCEGSRDDYYTPGNGPSYGTAPCFLDNEQRGCSYLFNYH SVKHFLLTNGLLCVVRSHEVQDDGYKLYRFNSVSNFPCMMSVFSAPNYCDNLHNKGAVLILEGKQIGIKQ FCCSPHPYVLPKHLNAFEWSFSYLLDSVRDIFASIISCEGA